J Med Dent Sci 2014； 61： 23－31

Original Article
Development of Oral and Extra-Oral Endosseous Craniofacial Implants by Using a
Mesh Structure for Connective Tissue Attachment
Atsushi Mita, Atsushi Yagihara, Wei Wang and Kazuo Takakuda

Medical and Dental Device Technology Incubation Center, Institute of Biomaterials and Bioengineering,Tokyo Medical and Dental University

Connective tissue attachment to a mesh structure
incorporated on the surface of oral implants
and extra-oral endosseous craniofacial implants(EOECI) was investigated. Two types of implants
were prepared: TI and TI-Mesh. TI was composed ofan upper and a lower component, both comprisedof a titanium cylinder, which could be connectedusing a titanium screw. The composition of the TIMesh was similar, but the lower cylinder had alateral groove that was covered with a titanium
mesh. In animal experiments performed using rat
calvaria, the lower component was first implanted
and was left submerged for 3 weeks, then theupper component was mounted percutaneously.
After an additional 2 weeks, each implant and thesurrounding tissues were harvested and evaluated.
Histological observations revealed collagen fibers
originating from surrounding hypodermal tissues
anchored to the mesh structures of the TI-Meshwhereas no such collagen fibers were observed
around TI. Significantly greater values of theattachment strength, the thickness of the dermal
tissue, the thickness of hypodermal tissue, and theattachment lengths were observed in TI-Mesh thanthose of TI. Thus connective tissue attachmentwith collagen fibers anchored to the mesh wasachieved by incorporating mesh structures into
the percutaneously placed implants.

Corresponding Author: Kazuo Takakuda
Medical and Dental Device Technology Incubation Center, Instituteof Biomaterials and Bioengineering, Tokyo Medical and Dental
University, 2-3-10 Kanda-Surugadai, Chiyoda-ku Tokyo 101-0062,
Japan
E-mail: takakuda.mech@tmd.ac.jp
Received September 30；Accepted November 8, 2013

Key words: Soft-tissue implant interactions, tissueimplant interface, connective tissue
attachment, animal experiments, mesh
Introduction
Oral implants and extra-oral endosseous craniofacial
implants (EOECI) are commonly used in clinical practice.
They are based on well-established technology of
1-4
and enable firm bonding of theosseointegration
implants to the bone tissues. However, the bonding of
implants to soft tissue has not yet been thoroughly
investigated.
At the interface between soft tissue and an implant,
the collagen fibers in the peri-implant tissues cannot
anchor to the implant, and run parallel to the implantsurface.5-8 With regard to natural teeth and the gingiva,
however, soft tissue attachment is established, whereby
the dento-gingival fibers from subepithelial connective
tissue run into the cementum of the root’s cervix andfirmly attach the gingiva to the tooth. Accordingly, thebond between oral implants and soft tissue is inferior
compared to that of natural teeth and surrounding soft
tissue owing to a lack of connective tissue attachment,
which leads to inferior marginal sealing and then topoor resistance against infection.9-11 Further, in the caseof EOECI, the lack of soft tissue attachment leads to thefar more serious clinical problem of downgrowth.12-15
Hence, improvement of the strength of attachment
between the implant and soft tissue is important inorder to overcome these problems. Many methods ofimproving the attachment of soft fibrous tissues to
artificial materials have been investigated. Among thesemethods, surface treatment processes such as coatingwith hydroxyapatite 16 or laminin-derived peptide, 17
micromachining of grooves,18 and laser ablation (LaserLok)19 have been reported to increase cell adhesion to24

A. Mita et al.

the surface of materials. However, attachment of theextracellular matrix to the surface of materials is
another issue that remains to be investigated.
We developed a mesh structure for spontaneousanchoring of collagen fibers originating from adjacentconnective tissues to the implant. Previous animal
experiments have shown that a mesh with a spacing ofapproximately 200 μm was favorable for soft tissue
attachment and for increasing attachment strength.20
However, the efficacy of the titanium implant with atitanium mesh structure has not been confirmed in
EOECI or oral implants models, in which the implant isplaced onto the bone percutaneously or permucosally.
In this study, implants with a mesh structure were
percutaneously anchored to rat calvaria, and theeffectiveness of the mesh structure was examined
under conditions approximating those of clinically usedimplants.

J Med Dent Sci

Figure 2. Experimental implants. The upper components were
mounted onto the lower components with a titanium screw.
(a) TI implant. (b) TI-Mesh implant incorporating a titanium meshcovering a groove on the lateral surface.

Materials and Methods
Experimental implants
The materials used for making experimental implants
were CP titanium rods and CP titanium meshes (Grade1, machined surface; Nilaco, Tokyo, Japan). Scanningelectron microscopic (SEM) images of these materials
are shown in Fig. 1. The dimensions of the titanium
mesh were determined using a measuring microscope(VH-8000; Keyence, Osaka, Japan); the fiber diameterand mesh spacing were found to be 120 μm and 213
μm, respectively.

Figure 1. Scanning electron microscopy (SEM) observations.
(a) Lateral surface of the titanium rod used for TI and TI-Mesh
implants. (b) Titanium mesh used for covering the groove of TI-Mesh
implants.

Two types of experimental implants were preparedin this study, which we called TI and TI-Mesh.
Photographs of these experimental implants are shownin Fig. 2, and their dimensions are presented in Fig. 3
in a schema representing implantation. The TI implant
was composed of upper and lower components. The
lower component was cylindrical, with a diameter of

Figure 3. A schema of implantation depicting the specimenʼsdimensions.
(a) First surgery. Implantation of the lower component alone. The
implant was fixed to the bone and left submerged for 3 weeks. (b)Second surgery. The upper component was mounted onto the lower
component using a titanium screw. After mounting, the upper
portion of the connected implant was left exposed for the following
2 weeks.

6 mm and a height of 3 mm, and it had a female M2screw hole on the upper surface. This screw hole
was used for mounting the upper component at thesecond surgery and for the connection of the jig at themechanical testing stage, as described later. The upper

Oral and EOEC Implants for Connective Tissue Attachment

component was also cylindrical, with outer and innerdiameters of 6 mm and 2 mm respectively and a heightof 2 mm. The TI-Mesh implant was similarly composedof 2 components, and the upper component was similar
to that of the TI implant. The lower component had thesame dimensions as that of the TI, but it had a lateralgroove of 1-mm width and 0.5-mm depth together withthe titanium mesh covering the whole lateral surface;
thus the lower component of TI-Mesh had the outer
diameter of 6.24 mm. Bonding of the mesh to thetitanium cylinder was achieved with the use of dental
adhesive (Super-Bond C&B; Sun medical, Shiga, Japan).
Under a stereomicroscope (SMZ1000; Nikon, Tokyo,
Japan), the mesh was adhered to the lateral surfaces
above and below the groove with paying particularattention not to allow the adhesive to overflow into thegroove. After hardening, the protruded adhesive overthe mesh was carefully removed with the use of dental
silicone point abrasives. These experimental implants
were degreased with acetone for 10 sec, rinsed with
70% ethanol and then with distilled water, dried at room
temperature, sterilized with ethylene oxide gas, andused for subsequent experiments.
Animal Experiments
Twenty 15-week-old male Sprague–Dawley rats (bodyweight: 500–600 g) were used in this study. The
experiment was conducted in compliance with theguidelines issued by the Institutional Animal Care and
Use Committee of Tokyo Medical and Dental University,and the study protocol was approved by the committee(Approval No. 0070176).
First, animals underwent implantation of the lower
component of 1 of the 2 types of implants. Before
surgery, the animal was initially anesthetized using
isoﬂurane (Isoﬂu; Abbott Lab., Abbott, IL, USA) and thenplaced under general anesthesia using medetomidine(0.5 mL/kg; Domitor; Pfizer AH, Exton, PA, USA)and sodium pentobarbital (0.5 mL/kg; Somnopentyl,
Schering-Plough AH, Omaha, NE, USA). Enrofloxacin(5 mg/kg; Baytril; Bayer AG, Leverkusen, Germany),
an antimicrobial agent, was also administeredintramuscularly to prevent infection. Then the animal’shead was shaved and disinfected with iodine solution,
and a straight incision was made extending from thenasal bone to the midsagittal crest. A periosteal flap
was elevated to expose the calvaria, in which a holeof 6-mm diameter and 0.8-mm depth was createdusing round and fissure dental burs under infusion
with physiological saline. After hemostasis, the bone
surface was washed with saline and air-dried. Then the25

dental adhesive was applied to the bottom surface of
the implant, and the implant was placed into the hole asshown in Fig. 4. The protruded adhesive was carefully
removed with a probe. Immediately after hardeningof the adhesive, hypodermal and dermal layers
were closed using 6-0 nylon sutures. After surgery,atipamezole hydrochloride (10 mL/kg; Antisedan;
Pfizer), a medetomidine antagonist, was administeredintramuscularly to reduce the recovery time fromanesthesia.
The implant was left submerged for 3 weeks, andthen the animal was subjected to a second surgery.
The soft tissue above the implanted lower component
was removed using a 6-mm-diameter soft tissue punch
to expose the top surface of the lower component, andthe upper component was mounted onto it with atitanium screw as shown in Fig. 4. The upper portion of
the connected implant was left exposed for 2 weeks.

Figure 4. Intraoperative photographs.
(a) First surgery. The lower component was inserted into the holeprepared on the calvaria and fixed via the application of a dental
adhesive. (b) Second surgery. The upper component was mounted
onto the lower component using a titanium screw. The upper
portion of the connected implant was left exposed.

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A. Mita et al.

Harvesting of samples
At the end of the implantation period, the implant was
retrieved along with the surrounding bone and skintissues, and mechanical tests and histologicalobservations were performed. Of the total 20 animals,
10 were used for mechanical testing (5 each for TI and
TI-Mesh implants) and the remaining 10 for histologicalobservation.
Mechanical testing
The harvested implant with the surrounding tissues
was immediately frozen at －24°C. Within a week,
preparation of the sample for mechanical testing was
performed without thawing, in which the bone tissues
adhering to the implants (the bone tissues beneath orlateral to the implants) were carefully removed under
the stereomicroscope using a dental round bur. Since
the bone tissues were of 0.1 - 0.2 mm thickness, it took
no more than several seconds to remove them. Then,the samples were soaked in physiological saline for 30
min and thawed. The attachment strengths of TI and TIMesh implants to the surrounding soft tissues were
measured by pull-out tests, in which a testing machine(Autograph AG-X; Shimadzu, Kyoto, Japan) was used as
illustrated in Fig. 5. The soft tissues and bone were
sandwiched between aluminum plates with a center
hole of 8 mm diameter and fixed to the lower crossheadof the testing machine. The upper component of theimplant was removed and the lower component wasconnected with a screw to a universal joint under theload cell fixed on the upper crosshead. The mechanicaltest was performed at a crosshead speed of 2 mm/min
and the data of load and displacement were recorded
via a computer. The area of the mesh facing the tissues
was adopted as the nominal area and calculated to be2
19.6 mm (1 mm [the height of the effective mesh] ×
3.14 × 6.24 mm [the diameter of the implant]); the peakload measured was divided by this nominal mesh area,
and the resulting value was determined as theattachment strength of TI-Mesh implant. The nominal
area of TI implant at the time of implantation was
similarly calculated to be 56.5 mm2 (3 mm [the height of
the titanium component] × 3.14 × 6.0 mm [thediameter of the implant]); however, considerable
downgrowth of skin tissues was observed in theexperiments and usage of this nominal area was foundto yield too small estimation for the strength. Observing
that the attachment length of connective tissues to TI
implant turned to be ca. 1 mm, the nominal area of TI
implant at the time of harvest was assumed to be 18.8mm2 (1 mm [the attachment length] × 3.14 × 6.0 mm

J Med Dent Sci[the diameter of the implant]) and the attachment
strength was evaluated. It should also be noted that thestatistical analysis demonstrated the same results on
estimated strengths independent of the utilized nominal
areas.

Figure 5. Schema of the mechanical testing of harvested
samples.

Histological observation
For histological observation, the experimental
implants and their surrounding tissues were fixedin 10% formalin immediately after harvest. They
were then dehydrated in a graded series of ethanol
dilutions, defatted with xylene, and embedded
in methylmethacrylate resin (Osteoresin; Wako,
Osaka, Japan). Thin sections of approximately 50μm thickness were prepared using a diamond discmicrotome (SP1600; Leica Microsystems, Bensheim,
Germany); these sections were stained with toluidine
blue and observed under a light microscope (Eclipse
50i; Nikon) equipped with a digital camera (Digital Sight
DS-SM5; Nikon). The collagen fibers in these sections
were also observed under polarized light.
Quantitative morphological evaluation of thehistological sections was performed. The areas of the21
adjacent tissues of 1.5-mm width around the implant
were specified in the histological section, and theaverage thickness of dermal and hypodermal tissues
were determined after dividing the measured area by
the width 1.5 mm. Attachment length was estimated asOral and EOEC Implants for Connective Tissue Attachment

27

the thickness of soft connective tissue (dermal tissue
plus hypodermal tissue) facing the implant surface.

attachment strength of TI-Mesh implants was
significantly greater than that of TI implants (p < 0.01).

Statistical analysis
The attachment strength of the implant to the soft
tissue, the thickness of the dermal tissue andhypodermal tissue in the surrounding soft tissue, andthe attachment length of the soft tissue to the implant
were statistically compared between TI and TI-Mesh
implants using the non-parametric Mann–Whitney U test.
All statistical analyses were performed using the “R”
software (version 2.13.0; http://www.r-project.org/).
Differences were considered statistically significant if p
< 0.05.

Histological observation
Typical histological sections stained with toluidine
blue are shown in Fig. 7. Significant downgrowth wasobserved in conjunction with the TI implants, whereas
none was evident with the TI-Mesh implants. Collagenfibers from the connective tissues surrounding TI-Mesh
implants entered into the groove as shown in Fig. 8, as
observed under polarized light. Collagen fibers
penetrated into the groove through the mesh and wereoriented perpendicular to the implant, and thus

Results
Macroscopic observation
During the experimental period, the condition of thebody, in general, and the tissue around the implant
were observed every day and no serious complications
such as infection were observed. At the time of theharvest, significant downgrowth was observed in all therats with TI implants, whereas none of the rats with TIMesh implants showed downgrowth.
Attachment strengths
The attachment strengths of both implants are shownin Fig. 6. The median (quartile range) of attachment
strength for TI-Mesh implant was 268 (194 – 317) kPa,
while that for TI implants 12 (12 – 24) kPa. The

Figure 6. Boxplot of Attachment strength of the implants tosoft connective tissues.
The bottom and top of the box are the first and third quartiles, theband inside the box is the median, and the ends of the whiskers are
the minimum and maximum of all of the data. The attachment
strength of TI-Mesh implants was significantly greater than that of
TI implants (p < 0.01).

Figure 7. Light microscopy observations (toluidine blue stain).
(a) TI implant. (b) TI-Mesh implant. D: dermis, H: hypodermis, B: bone,
Ti: titanium implant. The arrows in (a) and (b) indicate the length ofsoft connective tissue attachment. Cross sections of the titanium
mesh showed circular black spots (b).

Figure 8. The TI-Mesh implant shown in Fig. 6, as observed
under polarized light.
A magnified view of the box in (a) is shown in (b). Collagen fibers
were penetrating into the groove through the mesh and wereoriented perpendicular to the implant, and anchoring of theconnective tissues to the titanium mesh was confirmed. D: dermis, H:
hypodermis, B: bone, Ti: titanium implant, M: Ti mesh, CF: collagenfibers.

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J Med Dent Sci

A. Mita et al.

anchoring of the connective tissues to the titanium
mesh was achieved.
The results of quantitative morphological evaluationof the histological sections are shown in Fig. 9. The

thicknesses of dermal and hypodermal tissues were0.81 (0.58 – 1.09) mm and 0.78 (0.59 – 1.02) mm for TIMesh implants, and 0.34 (0.25 – 0.58) mm and 0.071(0.0 – 0.35) mm for TI implants respectively. The
attachment lengths were 1.74 (1.60 – 2.18) mm for TIMesh implants and 0.95 (0.77 – 1.20) mm for TI implants.
Significant differences were confirmed between eachpair of corresponding values derived from TI-Mesh and
TI implants (p < 0.01).
Discussion

Figure 9. Boxplot of (a) average thickness of dermal tissue, (b)average thickness of hypodermal tissue, and (c) attachment
length.
The bottom and top of the box are the first and third quartiles, theband inside the box is the median, and the ends of the whiskers are
the minimum and maximum of all of the data. Significant differenceswere confirmed for each of these values (p < 0.01).

Peri-implant downgrowth is one of the major clinicalconcerns relating to the long-term survival of
implants.22, 23 A probable cause of it is that the collagenfibers from the surrounding soft tissues are aligned
parallel to the material’s surface and are not anchoredto the implant.7, 19, 24 Therefore, it was expected that if
anchoring of collagen fibers from the surrounding soft
tissues to the surface of the implants was induced, thenenhanced attachment of fibrous connective tissue tothe implants could be achieved. A study investigating
the anchoring of collagen fibers to implants via the useof a mesh structure on the implant’s surface showedthat a mesh with approximately 200-μm spacing wasfavorable. 20 However, whether or not this meshstructure also effectively prevented peri-implant
downgrowth was not investigated. Hence, in this study,
downgrowth was examined with regard to titanium
implants that included a mesh structure and placed
percutaneously.
In this study, an experimental model was developed inwhich the specimen was implanted onto the rat calvaria
and the efficacy of the implant with mesh structures
was evaluated with regard to soft tissue attachment
and downgrowth. This kind of model has not beenreported elsewhere. The most technically difficult
aspect of this model was fixation of the implant to thecalvaria. Since this bone was as thin as 1 mm, theconventional technique of osseointegration1-4 was foundto be difficult to implement. Hence, a dental adhesive,
which had been proven to be biocompatible and usable
for bonding titanium implants to living bone tissues, 25
was employed, and it yielded successful fixation. We
believe this model is useful to investigate the efficacy
of connective tissue attachment to prevent theepithelial downgrowth. 19 However, the fact that
osseointegration was not achieved in this model was apotential limitation of this study.
Regarding the experimental period, Paquay et al .26
reported that acute inflammation due to surgery

Oral and EOEC Implants for Connective Tissue Attachment

continued for 2 weeks and the necessary healing
period was approximately 3 weeks, in their investigation
into experimental percutaneous devices fabricatedwith a sintered titanium fiber mesh. Therefore, in thisstudy, the lower components were left submerged for 3
weeks to allow the interface between the implant andthe hypodermal tissue to mature. Thereafter, the uppercomponents were mounted onto the lower components
in order to investigate downgrowth. A pilot experimentshowed that all of the TI implants had dislodged
approximately 3 weeks after the mounting of the uppercomponent. Hence, the experimental period after themounting of the upper component was determined to be
2 weeks.
The retrieved implants were evaluated with regard to
the attachment strength of soft tissue to the implant, theorientation of the collagen fibers, and the quantitative
morphological parameters of the soft tissues. The
space between the TI-Mesh implant and the aluminum
plate was smaller than that around TI implant. This
yielded the decrease in maximum bending displacementof the specimen. However, such displacement wasinherently small since the bending deformation of thebone tissue in the specimen was small. On the other
hand, the failure of the specimen was caused by theattachment failure of soft connective tissues after large
deformation. Hence the above mentioned differencein the spaces was considered to have relatively small
impact on the experimental results. The length ofdowngrowth may be defined as the sinking length ofthe epithelial tissue from the position of initial healing
after the surgery; however, such a position could not
be determined from the histological sections obtained
from the samples in which downgrowth had alreadyoccurred. Therefore, the attachment length of the soft
tissues to the implant surface was measured instead.
Greater attachment length is considered to correspondwith less downgrowth. Furthermore, since it is difficult
to strictly identify the location on the implant where
epithelium stopped, such histomorphological quantities
closely related to the attachment length as the
thickness of dermal tissue and hypodermal tissues werealso measured.
The mechanical attachment strength between TI-Mesh
implants and soft connective tissue was greater thanthat of TI implants. These results are consistent with the20, 27
Thus, it was verified
results of our previous studies.
that the mechanical attachment strength was increasedby using implants with the surface mesh structure, even
if it was placed onto the bone percutaneously.
Significant differences were found with regard to all29

the quantitative variables evaluated in the quantitative
morphological analyses (p < 0.01). The mesh structure
was considered to prevent the downgrowth; however, it
is possible to speculate that the difference in thesurfaces (titanium and the dental adhesive) was
responsible for the above mentioned differences.
Actually we performed a pilot study in which one-piece
implants (the implants which were not separated to theupper and lower components) were prepared andimplanted into the rat calvaria percutaneously. The
downgrowth was confirmed in either case of the Ti
surface (one-piece TI implant) or the dental adhesive
surface (one-piece TI-Mesh implant). In the case of the
one-piece TI-Mesh implant, the downgrowth took place
before the anchoring of collagen fibers to the mesh anddisabled the functionality of the mesh structurecompletely. Hence we concluded that the meshstructure effectively prevented the occurrence ofdowngrowth whereas the dental adhesive surface did
not.
While no studies on the downgrowth of the soft
tissues surrounding implants have been reported
elsewhere to date, the circumference of the implant in
the present study approximates that of EOECI. The
occurrence of downgrowth around our control TI
implants is consistent with the findings of clinical cases
12-14
On the other hand, such downgrowth wasof EOECI.
not observed in the experimental group implanted with
TI-Mesh. Downgrowth is far more frequently observedin the skin tissue around EOECI than in the masticatory
mucosa that surrounds an ordinary oral implant. Thisdifference may stem from differences between skintissue and masticatory mucosa. Skin tissue is relatively
soft and covering the soft hypodermal tissue andmuscle layer, thereby it shows high mobility. In contrast,
the masticatory mucosa is relatively hard and it islocated directly on hard bone tissue; thus it has lessm o b i l i t y . 28, 29 I n o r d e r t o r e d u c e t h e a m o u n t o fdowngrowth around EOECI, procedures in which theperi-implant soft tissue is thinned or the split thickness
skin is grafted (STSG technique) to limit tissue
movement are recommended. 30-33 In our results,
increased attachment strength achieved via theanchoring of collagen fibers to the mesh structure wasconsidered to have effectively decreased mobility andprevented downgrowth. Goldman24 and Nevins19 havealso asserted that connective tissue attachment of thecollagen fibers is the most important factor inpreventing downgrowth. In this study, the efficacy ofthe mesh structure for preventing downgrowth aroundthe implant was proven quantitatively, at least in a rat

30

A. Mita et al.

model of EOECI.
In fact, this experiment could be regarded as an
approximating model of EOECI, in which clinicians
experience many serious problems stem from the lackof adhesion of skin to implant, low stiffness of periimplant tissue, excessive thickness of peri-implanttissue, and mobility of peri-implant skin.15 The present
results suggested that an implant with a mesh structure
may help overcome these adverse events. However,further investigations concerning the long-term stabilityof the attachment and implantation studies relating tothe oral environment are necessary with regard toclinical applications.
Conclusions
This study showed that connective tissue attachment
via collagen fibers was achieved by incorporating amesh structure onto the experimental implant, even if
the implant was placed on the bone percutaneously.
This attachment enabled the dermal and hypodermal
tissue to remain healthy and prevented down-growth.
Implants with a mesh structure may be favorably
applied in clinical cases of EOECI and oral implantation.

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Acknowledgements
This study was partially supported by a Grant-in-Aid
for Scientific Research (B) No. 23300163 from the
Ministry of Education, Culture, and Sports, of Japan.

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